Application of combination of polyethylene glycol and local anesthetic in non-narcotic analgesia

Abstract

The invention discloses use of a conjugate of polyethylene glycol and a local anesthetic in non-anesthetic analgesia. A local anesthetic is prepared into a prodrug or a sustained release preparation, wherein a high molecular polymer such as polyethylene glycol in the prodrug is covalently bonded with a local anesthetic, and auxiliary materials with a sustained release effect in the sustained release preparation are non-covalently bonded to the local anesthetic. After administration, there is no anesthesia and analgesic effect before the release of the free local anesthetic. After the free local anesthetic is released, an analgesic effect is achieved. And the prodrug or the sustained-release preparation of the local anesthetic of the present invention releases the drug slowly, and renders the drug concentration kept stable and long-lasting in the effective concentration range of non-narcotic analgesia, and the long-acting non-anesthetic analgesic effect can be achieved while significantly reducing the clinical adverse reactions of local anesthetics and reducing the number of administrations. The effectiveness of the drug is greatly enhanced and the clinical application range of local anesthetics is expanded.

Claims

1. A method of using a local anesthetic release system for non-anesthetic analgesia treatment, the method comprising: administering the local anesthetic release system to a subject, the local anesthetic release system is a prodrug of a local anesthetic, wherein the prodrug of the local anesthetic is a conjugate of polyethylene glycol and a local anesthetic, having a structure of a formula (I):
PEG-A-R.sub.0—B   (I) wherein, PEG is a polyethylene glycol residue with a molecular weight of 1-100 KDa; R.sub.0 is a C.sub.1-6 alkyl group; B is a local anesthetic; a quaternary ammonium salt having a structure of ##STR00072##  is formed at the junction of B and R.sub.0, and R.sup.s0 is selected from a group consisting of F.sup.−, Cl.sup.−, Br.sup.−, I.sup.−, mesylate, ethylsulfonate, benzenesulfonate, citrate, lactate, succinate, fumarate, glutamate, citrate, salicylate and maleate; A is a linking group selected from a structure shown by the following formula A.sub.1 or A.sub.2: ##STR00073## wherein, R.sub.1 and R.sub.6 are independently selected from a C.sub.1-6 alkyl group, or R.sub.1 and R.sub.6 are independently selected from —(CH.sub.2).sub.iNHCO(CH.sub.2).sub.j—, or —(CH.sub.2).sub.iCONH(CH.sub.2).sub.j—, and i and j are independently an integer selected from 0 to 6; R.sub.2 is selected from —C═O, —C═S, —O— or —S—; R.sub.3 and R.sub.7 are independently selected from —O— or —S—; R.sub.4 and R.sub.5 are independently selected from H, C.sub.1-6 alkyl or halogen; R.sub.8 and R.sub.9 are independently selected from H, C.sub.1-6 alkyl or —O(C═O)(CH.sub.2).sub.iCH.sub.3, and i is an integer selected from 0 to 6; R.sup.s1 is selected from H or C.sub.1-6 alkyl.

2. The method according to claim 1, wherein the concentration of free local anesthetics released in the local tissue and/or systemic part is maintained between a minimum anesthetic dose and minimum analgesic dose of the local anesthetic after administration of the local anesthetic delivery system.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

(1) The technical solutions of the present invention will be described clearly and completely in conjunction with the examples of the present invention. It is obvious that the described examples are only a part of the examples of the present invention, and not all of the examples. All other examples obtained by those skilled in the art based on the examples of the present invention without creative efforts are within the scope of the present invention.

(2) The polyethylene glycol used in the examples was supplied by Beijing Jenkem Technology Co., Ltd., unless otherwise specified, the molecular weight was 20K. Others are commercially available reagents.

Example 1 Synthesis of Linking Chain (L1)

(3) ##STR00049##

(4) 4-Pentynoic acid (1.96 g, 20 mmol) and N,N-dicyclohexylcarbodiimide (DCC, 5.16 g, 25 mmol) are added to dichloromethane (50 mL), and cooled with an ice bath, then p-hydroxy benzaldehyde (2.68 g, 22 mmol) is added, after the addition is completed, the ice bath is removed and the mixture is allowed to react at room temperature overnight. After filtration, the filter cake is washed with ethyl acetate, and the filtrate is evaporated to dryness to obtain a crude product. The crude product is purified by column chromatography to yield 3.52 g of product 1a.

(5) The compound 1a (3.23 g, 16 mmol) is added anhydrous methanol (35 mL), and cooled to 0° C., then sodium borohydride (365 mg, 9.6 mmol) is added, and a reaction is carried out for 10 min at the same temperature, and then the reaction is quenched with 1 M HCl. The methanol is rotary-evaporated to dryness. The ethyl acetate and saturated brine are added. The aqueous phase is extracted with ethyl acetate, and the organic phases are mixed, washed with saturated brine, dried, filtered and concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 1.80 g of product 1b.

(6) Iodine (3.35 g, 13.2 mmol), triphenylphosphine (3.46 g, 13.2 mmol) and imidazole (0.90 g, 13.2 mmol) are added in dichloromethane (30 mL), and stirred at 0° C. for 20 min, a solution of the product 1b (1.80 g, 8.8 mmol) in dichloromethane (7.5 mL) is added and a reaction is carried out at 0° C. for 30 min. The liquid mixture is washed with 2N hydrochloric acid, saturated sodium bisulfite and brine respectively, dried and evaporated to dryness to obtain a crude product. The crude product is purified by column chromatography to yield 2.41 g of product L1. .sup.1H NMR: (CDCl.sub.3): 2.03 (s, 1H), 2.61 (t, 2H), 2.77 (t, 2H), 4.44 (s, 2H), 7.01 (d, 2H), 7.39 (d, 2H).

Example 2 Synthesis of Lidocaine Quaternary Ammonium Salt (Y1)

(7) ##STR00050##

(8) Lidocaine (0.50 g, 2.13 mmol) and the compound L1 (1.00 g, 3.19 mmol) are added to acetonitrile (20 mL) and reacted at 50° C. overnight. TLC monitoring showed that the lidocaine is completely reacted and the reaction solution is concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 1.19 g of product Y1. .sup.1H NMR: (CDCl.sub.3): 1.59 (m, 6H), 2.06 (s, 2H), 2.31 (s, 6H), 2.64 (m, 2H), 2.87 (m, 2H), 3.49 (m, 2H), 3.71 (m, 2H), 4.89 (s, 2H), 4.93 (s, 2H), 7.06 (m, 3H), 7.28 (d, 2H), 7.62 (d, 2H), 9.79 (s, 1H).

Example 3 Synthesis of Lidocaine Conjugate 1 (mPEG-Lidocaine, 20K)

(9) ##STR00051##

(10) mPEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y1 (65.8 mg, 0.12 mmol), and vitamin C (52.8 mg, 0.30 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. Then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (30.0 mg, 0.12 mmol) is added, and the mixture is reacted overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.90 g of product. .sup.1H NMR: (CDCl.sub.3): 1.42 (m, 6H), 2.19 (s, 6H), 3.02 (m, 4H), 3.23 (m, 4H), 3.31 (s, 3H), 3.50 (m, 1800H), 3.80 (m, 2H), 4.20 (m, 2H), 4.50 (s, 2H), 4.82 (s, 2H), 7.12 (m, 3H), 7.30 (d, 2H), 7.64 (d, 2H), 7.91 (s, 1H), 10.28 (s, 1H).

Example 4 Synthesis of Lidocaine Conjugate 2 (4-Arm-PEG-Lidocaine, 10K)

(11) ##STR00052##

(12) 4-arm-PEG-N.sub.3 (10K, 2.00 g, 0.20 mmol), the compound Y1 (548 mg, 1.00 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is reacted overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.83 g of product. .sup.1H NMR: (CDCl.sub.3): 1.43 (m, 24H), 2.22 (s, 24H), 3.07 (m, 16H), 3.25 (m, 16H), 3.34 (s, 12H), 3.50 (m, 900H), 3.83 (m, 8H), 4.22 (m, 8H), 4.53 (s, 8H), 4.85 (s, 8H), 7.13 (m, 12H), 7.30 (d, 8H), 7.65 (d, 8H), 7.92 (s, 4H), 10.26 (s, 4H).

Example 5 Synthesis of Lidocaine Conjugate 3 (8-Arm-PEG-Lidocaine, 20K)

(13) ##STR00053##

(14) 8-arm-PEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y1 (548 mg, 1.00 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is reacted at room temperature overnight, and precipitation is carried out with isopropyl alcohol to obtain 1.62 g of product. .sup.1H NMR: (CDCl.sub.3): 1.44 (m, 48H), 2.22 (s, 48H), 3.09 (m, 32H), 3.23 (m, 32H), 3.37 (s, 24H), 3.51 (m, 1800H), 3.84 (m, 16H), 4.23 (m, 16H), 4.54 (s, 16H), 4.86 (s, 16H), 7.12 (m, 24H), 7.31 (d, 16H), 7.66 (d, 16H), 7.93 (s, 8H), 10.27 (s, 8H).

Example 6 Synthesis of Bupivacaine Quaternary Ammonium Salt (Y2)

(15) ##STR00054##

(16) Bupivacaine (0.50 g, 1.74 mmol) and the compound L1 (1.00 g, 3.19 mmol) are added to acetonitrile (20 mL) and reacted at 50° C. overnight. TLC monitoring showed that the bupivacaine is completely reacted and the reaction solution is concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 0.90 g of product Y2. .sup.1H NMR: (CDCl.sub.3): 1.09 (m, 3H), 1.46 (m, 3H), 1.85 (m, 4H), 2.19 (m, 1H), 2.30 (s, 6H), 2.61 (m, 2H), 2.76 (m, 2H), 2.93 (s, 1H), 3.30 (m, 2H), 3.39 (m, 2H), 3.71 (m, 2H), 4.91 (s, 2H), 4.96 (s, 2H), 7.18 (m, 3H), 7.26 (d, 2H), 7.42 (d, 2H), 10.15 (s, 1H).

Example 7 Synthesis of Bupivacaine Conjugate 4 (mPEG-Bupivacaine, 20K)

(17) ##STR00055##

(18) mPEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y2 (89.7 mg, 0.15 mmol), and vitamin C (52.8 mg, 0.30 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (30.0 mg, 0.12 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.72 g of product. .sup.1H NMR: (CDCl.sub.3): 1.09 (m, 3H), 1.25 (m, 1H), 1.37 (m, 3H), 1.75 (m, 4H), 1.97 (m, 1H), 2.16 (s, 6H), 2.23 (m, 1H), 2.57 (m, 2H), 2.85 (m, 2H), 3.27 (m, 4H), 3.32 (s, 3H), 3.56 (m, 1800H), 3.95 (m, 2H), 4.10 (m, 2H), 4.52 (s, 2H), 4.60 (s, 2H), 7.19 (m, 3H), 7.27 (d, 2H), 7.30 (d, 2H), 7.69 (s, 1H), 10.19 (s, 1H).

Example 8 Synthesis of Bupivacaine Conjugate 5 (4-Arm-PEG-Bupivacaine, 10K)

(19) ##STR00056##

(20) 4-arm-PEG-N.sub.3 (10K, 2.00 g, 0.20 mmol), the compound Y2 (723 mg, 1.20 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250.0 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.82 g of product. .sup.1H NMR: (CDCl.sub.3): 1.08 (m, 12H), 1.26 (m, 4H), 1.38 (m, 12H), 1.74 (m, 8H), 1.98 (m, 4H), 2.17 (s, 24H), 2.25 (m, 4H), 2.56 (m, 8H), 2.83 (m, 8H), 3.27 (m, 16H), 3.31 (s, 12H), 3.54 (m, 900H), 3.95 (m, 8H), 4.07 (m, 8H), 4.50 (s, 8H), 4.58 (s, 8H), 7.21 (m, 12H), 7.29 (d, 8H), 7.33 (d, 8H), 7.65 (s, 4H), 10.15 (s, 4H).

Example 9 Synthesis of Bupivacaine Conjugate 6 (8-Arm-PEG-Bupivacaine, 20K)

(21) ##STR00057##

(22) 8-arm-PEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y2 (723 mg, 1.20 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.64 g of product. .sup.1H NMR: (CDCl.sub.3): 1.09 (m, 24H), 1.26 (m, 8H), 1.39 (m, 24H), 1.73 (m, 16H), 1.97 (m, 8H), 2.17 (s, 48H), 2.24 (m, 8H), 2.57 (m, 16H), 2.81 (m, 16H), 3.27 (m, 32H), 3.32 (s, 24H), 3.51 (m, 1800H), 3.93 (m, 16H), 4.09 (m, 16H), 4.51 (s, 16H), 4.57 (s, 16H), 7.20 (m, 24H), 7.27 (d, 16H), 7.32 (d, 16H), 7.63 (s, 8H), 10.16 (s, 8H).

Example 10 Synthesis of Linked Linking Chain L2

(23) ##STR00058##

(24) 3,4-dihydroxybenzaldehyde (25.0 g, 181 mmol) is added to N,N-dimethylformamide (180 mL), and cooled to 0° C., and sodium hydride (7.2 g, 181 mmol) is added in batches. After addition, the mixture is reacted for 10 minutes, and the temperature is further raised to room temperature. Acetic anhydride (18.7 g, 183 mmol) is added, and after addition, the mixture is reacted for 4 hours. Ice water and 10% hydrochloric acid are added to the reaction liquid, and extraction is carried out with ethyl acetate. The organic phases are mixed, washed with a saturated sodium bicarbonate solution, water and saturated brine respectively and dried. After concentration of the solution, the residue is crystallized by chloroform to obtain 19.8 g of product 2a.

(25) 3-Hydroxy-4-acetoxybenzaldehyde (5.40 g, 30 mmol) and sodium carbonate (9.54 g, 90 mmol) are added to N,N-dimethylformamide (50 mL) and heated to 60° C. for reaction for half an hour. Then the reacted material is cooled to room temperature, and a toluene (80%, 4.1 mL) solution of propargyl bromide is added. After the addition is completed, the reaction is continued to be carried out at the same temperature. After the reaction is completed, the mixture is poured into ice water, and the obtained solution is extracted with diethyl ether. The organic phases are mixed, washed with water and dried. After concentration, 5.32 g of product 2b is obtained.

(26) The compound 2b (4.36 g, 20 mmol) is added to absolute methanol (50 mL), and cooled to 0° C., then sodium borohydride (456 mg, 12 mmol) is added, the mixture is reacted for 10 min at the same temperature, and the reaction is quenched with 1 M HCl. The methanol is rotary-evaporated to dryness. The ethyl acetate and saturated brine are added. And the aqueous phase is extracted with ethyl acetate. The organic phases are mixed, washed with saturated brine, dried, filtered and concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 3.30 g of product 2c.

(27) Iodine (3.81 g, 15 mmol), triphenylphosphine (3.93 g, 15 mmol) and imidazole (1.02 g, 15 mmol) are added to dichloromethane (30 mL) and stirred at 0° C. for 20 min. A dichloromethane (10 mL) solution of the product 2c (2.20 g, 10 mmol) is added, and the mixture is reacted at 0° C. for 30 min. The liquid mixture is washed with 2N hydrochloric acid, a saturated sodium bisulfite solution and brine, dried and evaporated to dryness to obtain a crude product. The crude product is purified by column chromatography to yield 2.40 g of product L2. .sup.1H NMR: (CDCl.sub.3): 2.35 (s, 3H), 3.37 (s, 1H), 4.51 (s, 2H), 4.72 (s, 2H), 6.77 (d, 1H), 7.02 (s, 1H), 7.09 (d, 1H).

Example 11 Synthesis of Lidocaine Quaternary Ammonium Salt Y3

(28) ##STR00059##

(29) Lidocaine (0.50 g, 2.13 mmol) and the compound L2 (1.08 g, 3.27 mmol) are added to acetonitrile (20 mL) and reacted at 50° C. overnight. TLC monitoring showed that the lidocaine is completely reacted and the reaction solution is concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 0.71 g of product Y3. .sup.1H NMR: (CDCl.sub.3): 1.05 (m, 6H), 2.13 (s, 2H), 2.30 (s, 3H), 2.42 (m, 4H), 3.31 (s, 2H), 3.35 (s, 1H), 3.63 (m, 2H), 4.71 (s, 2H), 6.92 (d, 1H), 7.08 (d, 1H), 7.12 (s, 1H), 7.19 (m, 3H), 9.82 (s, 1H).

Example 12 Synthesis of Lidocaine Conjugate 7 (4-Arm-PEG-Lidocaine, 10K)

(30) ##STR00060##

(31) 4-arm-PEG-N.sub.3 (10K, 2.00 g, 0.20 mmol), the compound Y3 (677 mg, 1.20 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.83 g of product. .sup.1H NMR: (CDCl.sub.3): 1.05 (m, 24H), 2.13 (s, 24H), 2.30 (s, 12H), 2.42 (m, 16H), 3.50 (s, 8H), 3.53 (m, 900H), 3.86 (m, 8H), 3.91 (m, 8H), 4.55 (s, 8H), 4.81 (s, 8H), 5.23 (s, 8H), 6.91 (d, 4H), 7.06 (s, 4H), 7.20 (m, 12H), 7.68 (s, 4H), 10.26 (s, 4H).

Example 13 Synthesis of Lidocaine Conjugate 8 (8-Arm-PEG-Lidocaine, 20K)

(32) ##STR00061##

(33) 8-arm-PEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y3 (677 mg, 1.20 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.70 g of product. .sup.1H NMR: (CDCl.sub.3): 1.07 (m, 48H), 2.14 (s, 48H), 2.31 (s, 24H), 2.41 (m, 32H), 3.52 (s, 16H), 3.54 (m, 1800H), 3.79 (m, 16H), 3.92 (m, 16H), 4.53 (s, 16H), 4.82 (s, 16H), 5.22 (s, 16H), 6.90 (d, 8H), 7.09 (s, 8H), 7.28 (m, 24H), 7.69 (s, 8H), 10.27 (s, 8H).

Example 14 Synthesis of Bupivacaine Quaternary Ammonium Salt (Y4)

(34) ##STR00062##

(35) Bupivacaine (0.50 g, 1.73 mmol) and the compound L2 (1.08 g, 3.27 mmol) are added to acetonitrile (20 mL) and reacted at 50° C. overnight. TLC monitoring showed that the bupivacaine is completely reacted and the reaction solution is concentrated to obtain a crude product. The crude product is purified by column chromatogram to yield 0.75 g of product Y4. .sup.1H NMR: (CDCl.sub.3): 1.09 (m, 3H), 1.25 (m, 1H), 1.33 (m, 3H), 1.72 (m, 3H), 1.95 (m, 1H), 2.13 (s, 6H), 2.20 (m, 1H), 2.30 (s, 6H), 3.20 (m, 1H), 3.25 (m, 2H), 3.29 (m, 1H), 3.34 (s, 1H), 4.57 (s, 2H), 4.69 (s, 2H), 4.75 (s, 2H), 6.90 (d, 1H), 7.05 (d, 1H), 7.09 (d, 1H), 7.18 (m, 3H), 10.07 (s, 1H).

Example 15 Synthesis of Bupivacaine Conjugate 9 (4-Arm-PEG-Bupivacaine, 10K)

(36) ##STR00063##

(37) 4-arm-PEG-N.sub.3 (10K, 2.00 g, 0.20 mmol), the compound Y4 (748 mg, 1.20 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.82 g of product. .sup.1H NMR: (CDCl.sub.3): 1.09 (m, 12H), 1.25 (m, 4H), 1.33 (m, 12H), 1.72 (m, 12H), 1.95 (m, 4H), 2.13 (s, 24H), 2.20 (m, 4H), 2.30 (s, 24H), 3.20 (m, 4H), 3.25 (m, 8H), 3.28 (m, 4H), 3.50 (s, 8H), 3.54 (m, 900H), 3.86 (m, 8H), 3.91 (m, 8H), 4.52 (s, 8H), 4.59 (s, 8H), 5.23 (s, 8H), 6.82 (d, 4H), 7.03 (d, 4H), 7.09 (s, 4H), 7.18 (m, 12H), 7.65 (s, 4H), 10.13 (s, 4H).

Example 16 Synthesis of Bupivacaine Conjugate 10 (8-Arm-PEG-Bupivacaine, 20K)

(38) ##STR00064##

(39) 8-arm-PEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y4 (748 mg, 1.21 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.61 g of product. .sup.1H NMR: (CDCl.sub.3): 1.08 (m, 24H), 1.24 (m, 8H), 1.34 (m, 24H), 1.73 (m, 24H), 1.93 (m, 8H), 2.14 (s, 48H), 2.21 (m, 8H), 2.31 (s, 48H), 3.21 (m, 8H), 3.25 (m, 16H), 3.28 (m, 8H), 3.50 (s, 16H), 3.53 (m, 1800H), 3.85 (m, 16H), 3.92 (m, 16H), 4.53 (s, 16H), 4.59 (s, 16H), 5.22 (s, 16H), 6.81 (d, 8H), 7.05 (d, 8H), 7.09 (s, 8H), 7.19 (m, 24H), 7.67 (s, 8H), 10.12 (s, 8H).

Example 17 Synthesis of Linking Chain (L3)

(40) ##STR00065## ##STR00066##

(41) An aqueous formaldehyde solution (37% m/v, 28 mL, 375 mmol), an aqueous dimethylamine solution (40% m/v, 48 mL, 375 mmol) and absolute ethanol (250 mL) are added to a three-necked flask. 4-Hydroxy-3-methoxybenzaldehyde (38.9 g, 250 mmol) is added with stirring, and the mixture is heated to reflux and stirred for 45 minutes, then cooled to room temperature and stirred overnight. After filtration, the filter cake is washed with ice cold acetone and dried at room temperature under reduced pressure to yield 39.1 g of product 3a.

(42) 3a (21.0 g, 100 mmol) is dissolved in acetic anhydride (100 mL), and the mixture is heated to reflux under a nitrogen atmosphere and stirred for 24 hours. The reaction mixture is concentrated under reduced pressure. The residue is diluted with HCl (37% m/v, 100 mL) and stirred at room temperature for 2 hours. 1,4-Dioxane (100 mL) and stannous chloride dihydrate (69.8 g, 300 mmol) are added, and the mixture is heated to reflux and stirred for 45 minutes. After cooling to room temperature, hydrochloric acid (37% m/v, 20 mL) is added and then extraction is carried out with dichloromethane (5×100 mL). The mixed extract is washed successively with 6N hydrochloric acid, distilled water and an aqueous sodium chloride solution of 10%, and dried over anhydrous sodium sulfate, and a solvent is evaporated under reduced pressure. The residue is separated and purified with a silica gel column to yield 11.2 g of product 3b. 3b (8.35 g, 50 mmol) is dissolved in dichloromethane (200 mL), stirred and cooled to 0° C., then a solution of boron tribromide in dichloromethane (1M, 150 mmol, 150 mL) is slowly added dropwise under the protection of nitrogen. After adding dropwise, stirring is continued to be performed at 0° C. for 1 hour, and then the temperature is raised to room temperature and stirring is carried out overnight. After cooled to 0° C., the stirred material is diluted by slowly adding distilled water (350 mL), and the organic solvent is evaporated under reduced pressure, and after cooling to room temperature, filtering is carried out. The filter cake is washed with distilled water, transferred to a dry box and dried under reduced pressure to obtain a crude product. The crude product is separated and purified by using silica gel column to yield 5.73 g of product 3c.

(43) 3c (27.5 g, 181 mmol) is added to N,N-dimethylformamide (180 mL), cooled to 0° C., sodium hydride (7.2 g, 181 mmol) is added in batches, and a reaction is carried out for 10 minutes. After heating to room temperature, acetic anhydride (18.7 g, 183 mmol) is added, and a reaction is carried out for 4 hours. Ice water and 10% hydrochloric acid are added to the reaction liquid, and the reaction liquid is extracted with ethyl acetate. The organic phases are mixed, washed with a saturated sodium bicarbonate solution, water and saturated brine respectively and dried. After the solution is concentrated, the residue is crystallized with chloroform to yield 19.3 g of product 3d.

(44) 3d (17.5 g, 90 mmol) and sodium carbonate (28.6 g, 270 mmol) are added to N,N-dimethylformamide (150 mL), heated to 60° C. for reaction for half an hour, and then cooled to room temperature, and 5-bromopent-1-yne (19.8 g, 135 mmol) is added, and the reaction is continued to be carried out at the same temperature. After the completion of the reaction, the mixture is poured into ice water, and the resulting solution is extracted with diethyl ether. The organic phases are mixed, washed with water and dried. After concentration, 15.9 g of product 3e is obtained.

(45) 3e (15.6 g, 60 mmol) is added to absolute methanol (150 mL), and cooled to 0° C., and then sodium borohydride (1.82 g, 48 mmol) is added. The reaction is carried out at the same temperature for 10 min, quenched with 1 M HCl. The methanol is rotary-evaporated to dryness. The ethyl acetate and saturated brine are added. And the aqueous phase is extracted with ethyl acetate. The organic phases are mixed, washed with saturated brine and dried. After filtration, the filtrate is concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 10.5 g of product 3f.

(46) Iodine (11.4 g, 45 mmol), triphenylphosphine (11.8 g, 45 mmol) and imidazole (3.06 g, 45 mmol) are added to dichloromethane (90 mL) and stirred at 0° C. for 20 min, a solution of 3f (7.87 g, 30 mmol) in dichloromethane (30 mL) is added, and the mixture is reacted at 0° C. for 30 min. The liquid mixture is washed with 2N hydrochloric acid, a saturated sodium bisulfite solution and brine, dried and evaporated to dryness to obtain a crude product. The crude product is purified by column chromatography to yield 7.42 g of product L3. .sup.1H NMR: (DMSO-d.sub.6): 1.79-1.90 (m, 2H), 2.06 (s, 3H), 2.24-2.31 (m, 5H), 2.81 (m, 1H), 3.90-4.08 (m, 2H), 4.57 (s, 2H), 6.92 (d, 1H), 7.02 (d, 1H).

Example 18 Synthesis of Bupivacaine Quaternary Ammonium Salt (Y5)

(47) ##STR00067##

(48) Bupivacaine (2.50 g, 8.67 mmol) and L3 (1.19 g, 3.19 mmol) are added to acetonitrile (100 mL) and reacted at 50° C. overnight. TLC monitoring showed that the bupivacaine is completely reacted and the reaction solution is concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 4.51 g of product Y4. .sup.1H NMR: (CDCl.sub.3): 0.95-0.99 (m, 3H), 1.23-1.36 (m, 2H), 1.77-2.49 (m, 25H), 3.32-3.38 (m, 4H), 4.03-4.13 (m, 2H), 4.75-4.79 (s, 2H), 5.90-5.94 (m, 1H), 6.87 (s, 2H), 7.04-7.14 (m, 3H), 10.02 (s, 1H).

Example 19 Synthesis of Bupivacaine Conjugate 11 (8-Arm-PEG-Bupivacaine, 20K)

(49) ##STR00068##

(50) 8-arm-PEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y5 (793 mg, 1.20 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.60 g of product. .sup.1H NMR: (CDCl.sub.3): 0.95-0.99 (m, 3H), 1.23-1.36 (m, 2H), 1.77-2.49 (m, 22H), 2.82 (m, 2H), 3.32-3.38 (m, 4H), 3.53 (m, 1800H), 3.87-3.96 (m, 4H), 4.03-4.13 (m, 2H), 4.75-4.79 (s, 2H), 5.90-5.94 (m, 1H), 6.87 (s, 2H), 7.04-7.14 (m, 3H), 10.02 (s, 1H).

Example 20 Synthesis of Linking Chain (L4)

(51) ##STR00069##

(52) 3c (27.5 g, 181 mmol) is added to N,N-dimethylformamide (180 mL), cooled to 0° C., sodium hydride (7.2 g, 181 mmol) is added in batches, and the reaction is carried out for 10 minutes after the addition. After heating to room temperature, butyric anhydride (28.9 g, 183 mmol) is added, and the reaction is carried out for 4 hours after the addition. Ice water and 10% hydrochloric acid are added to the reaction liquid, and the reaction liquid is extracted with ethyl acetate. The organic phases are mixed, washed with a saturated sodium bicarbonate solution, water and saturated brine respectively and dried. After the solution is concentrated, the residue is crystallized with chloroform to yield 21.5 g of product 4d.

(53) 4d (20.0 g, 90 mmol) and sodium carbonate (28.6 g, 270 mmol) are added to N,N-dimethylformamide (150 mL), heated to 60° C. for reaction for half an hour, and then cooled to room temperature, 5-bromopent-1-yne (19.8 g, 135 mmol) is added, and the reaction is continued to be carried out at the same temperature. After the completion of the reaction, the mixture is poured into ice water, and the resulting solution is extracted with diethyl ether. The organic phases are mixed, washed with water, and dried. After concentration, 17.7 g of product 4e is obtained.

(54) 4e (17.3 g, 60 mmol) is added to absolute methanol (150 mL), and cooled to 0° C., and then sodium borohydride (1.82 g, 48 mmol) is added. A reaction is carried out at the same temperature for 10 min, and quenched with 1 M HCl. The methanol is rotary-evaporated to dryness. The ethyl acetate and saturated brine are added. And the aqueous phase is extracted with ethyl acetate. The organic phases are mixed, washed with saturated brine and dried. After filtration, the filtrate is concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 11.2 g of product 4f.

(55) Iodine (11.4 g, 45 mmol), triphenylphosphine (11.8 g, 45 mmol) and imidazole (3.06 g, 45 mmol) are added to dichloromethane (90 mL) and stirred at 0° C. for 20 min, a solution of 4f (8.7 g, 30 mmol) in dichloromethane (30 mL) is added, and the mixture is reacted at 0° C. for 30 min. The liquid mixture is washed with 2N hydrochloric acid, a saturated sodium bisulfite solution and brine, dried and evaporated to dryness to obtain a crude product. The crude product is purified by column chromatography to yield 7.8 g of product IA. .sup.1H NMR: (DMSO-d.sub.6): 0.98 (s, 3H), 1.78 (m, 2H), 2.06 (s, 3H), 2.23 (m, 2H), 2.24-2.31 (m, 5H), 2.81 (m, 1H), 3.90-4.08 (m, 2H), 4.57 (s, 2H), 6.92 (d, 1H), 7.02 (d, 1H).

Example 21 Synthesis of Bupivacaine Quaternary Ammonium Salt (Y6)

(56) ##STR00070##

(57) Bupivacaine (2.50 g, 8.67 mmol) and L4 (1.28 g, 3.19 mmol) are added to acetonitrile (100 mL) and reacted at 50° C. overnight. TLC monitoring showed that the bupivacaine is completely reacted and the reaction solution is concentrated to obtain a crude product. The crude product is purified by column chromatography to yield 4.82 g of product Y6. .sup.1H NMR: (CDCl.sub.3): 0.95-0.99 (m, 6H), 1.23-1.36 (m, 4H), 1.77-2.49 (m, 25H), 3.32-3.38 (m, 4H), 4.03-4.13 (m, 2H), 4.75-4.79 (m, 3H), 5.90-5.94 (m, 1H), 6.87 (s, 2H), 7.04-7.14 (m, 3H), 10.02 (s, 1H).

Example 22 Synthesis of Bupivacaine Conjugate 12 (8-Arm-PEG-Bupivacaine, 20K)

(58) ##STR00071##

(59) 8-arm-PEG-N.sub.3 (20K, 2.00 g, 0.10 mmol), the compound Y6 (826 mg, 1.20 mmol), and vitamin C (440 mg, 2.50 mmol) are added to N,N-dimethylformamide (20 mL), and stirred rapidly to make the above materials dissolved. And then an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1.00 mmol) is added, and the mixture is allowed to react overnight at room temperature, and precipitation is carried out with isopropyl alcohol to obtain 1.72 g of product. .sup.1H NMR: (CDCl.sub.3): 0.95-0.99 (m, 48H), 1.23-1.36 (m, 32H), 1.77-2.49 (m, 176H), 2.82 (m, 16H), 3.32-3.38 (m, 32H), 3.53 (m, 1800H), 3.87-3.96 (m, 32H), 4.03-4.13 (m, 16H), 4.75-4.79 (m, 16H), 5.90-5.94 (m, 8H), 6.87 (s, 16H), 7.04-7.14 (m, 24H), 10.02 (s, 8H).

Example 23 Bupivacaine Sustained Release Lyophilized Powder Preparation

(60) The main components and mass ratios are as follows:

(61) TABLE-US-00001 Bupivacaine hydrochloride 8 parts Hydrogenated soybean lecithin 6-6.5 parts Cholesterol 3 parts Glycerol trilaurate 0.1-0.2 part

(62) Hydrogenated soybean lecithin, cholesterol, and glycerol trilaurate are dissolved in chloroform as a lipid phase;

(63) Bupivacaine hydrochloride is dissolved in water as an aqueous phase;

(64) The aqueous phase is added to the lipid phase and shearing is carried out at a high speed to form a water-in-oil emulsion;

(65) The solvent is removed, and filtering, washing, and lyophilizing are carried out to obtain a liposomal lyophilized powder preparation of bupivacaine.

Example 24 In Situ Lipid Gel Preparation of Bupivacaine

(66) The main ingredients and dosage are as follows:

(67) TABLE-US-00002 Bupivacaine hydrochloride 50 mg/g the total weight of preparation Soybean Lecithin 40 ml Absolute ethanol 20 ml Water for injection 30 ml

(68) The above ingredients are mixed, placed in a sealed container, heated under magnetic stirring, fully dissolved, and cooled to obtain an in situ lipid gel preparation of bupivacaine.

Example 25 Sustained Release Tablets of Bupivacaine

(69) The main ingredients and dosage are as follows:

(70) TABLE-US-00003 Bupivacaine hydrochloride 5 mg Hydroxypropyl methylcellulose 50 mg Lactose 20 mg Magnesium stearate 10 mg Microcrystalline cellulose 30 mg

(71) Bupivacaine hydrochloride is mixed with microcrystalline cellulose, and hydroxypropyl methylcellulose, and lactose are added and the materials are mixed uniformly, the mixture is dissolved in ethanol to be wetted to make a soft material, the soft material is granulated, the granulated material is dried, granule dispersion is carried out, and magnesium stearate is added, the materials are mixed uniformly, and the mixture is tableted to obtain skeleton-dispersed sustained-release tablets of bupivacaine.

Example 26 Sustained Release Tablets of Bupivacaine

(72) The main ingredients and dosage are as follows:

(73) TABLE-US-00004 Cores of the tablets: Bupivacaine hydrochloride 5 mg Lactose 40 mg Ethyl cellulose 10 mg Magnesium stearate 20 mg Coating solution: Cellulose acetate 30 mg PEG 400 15 ml Acetone 100 ml

(74) Bupivacaine hydrochloride is uniformly mixed with lactose, ethyl cellulose in ethanol is added, so that a soft material is made, the soft material is sieved, wet granules are made, dried, and sieved, and magnesium stearate is added, the materials are mixed uniformly and the mixture is tableted. An acetone aqueous solution containing PEG 400 and cellulose acetate is used for spray-coating, and drying, and laser drilling are carried out to obtain membrane-controlled sustained-release tablets of bupivacaine.

Example 27 Bupivacaine Sustained Release Pills

(75) The main ingredients and dosage are as follows:

(76) TABLE-US-00005 Cores of the pills: Bupivacaine hydrochloride 80 mg Starch 40 mg Dextrin 45 mg Coating solution: Acrylic resin No. II 1.5 g PEG 400 3 ml Diethyl phthalate 0.01 g Talc powder moderate amount Ethanol adding up to 25 ml

(77) The bupivacaine hydrochloride is thoroughly and uniformly mixed with starch and dextrin, and the pellet core is made in a coating pot with a certain concentration of ethanol, taken out, and dried. After the fine powder is removed, the dried pellet core is weighed, and put in the coating pot to be coated to obtain bupivacaine sustained-release pellets. It can be further processed into other dosage forms.

Example 28 Bupivacaine Analgesic Minimum and Maximum Dose (or Minimum Anesthetic Dose) Test

(78) 1. Experimental Materials

(79) 1.1 Experimental animals: SD rats, SPF grade, male, 6-9 weeks old, 200-260 g, purchased from Shanghai Slack Laboratory Animals Co., Ltd., experimental animal certificate number: 2015000527734.

(80) 1.2 Experimental animal feeding conditions: The animal room environment is maintained at a temperature of 23±2° C., and a humidity of 40-70%, alternating 12 hours of light and dark. Experimental animals are acclimated for at least 7 days prior to the experiment.

(81) 1.3 Test substance: bupivacaine injection, diluted with physiological saline to the target concentration; the reference substance is physiological saline.

(82) 2. Experimental Method

(83) 2.1 Catheterization Around the Sciatic Nerve:

(84) After the animals are anesthetized by intraperitoneal injection of 10% chloral hydrate solution, the sciatic nerve is exposed to the biceps and gluteus maximus joints of the hind limbs through surgery, and silicone flexible catheters of the same length (7 cm) are placed around the sciatic nerve. the other ends of the catheters are subcutaneously embedded in the back of the rat, and then sutured and fixed, and 0.2 mL of an antibiotic solution (5,000 U/ml penicillin G) is injected for anti-inflammatory.

(85) 2.2 Screening of Animals:

(86) After 2-3 days of catheterization, the SD rats with the body weight of 230-250 g and into which catheters are embedded are screened before the experiment by a hot plate method. The hot plate is heated to (56±1)° C., one of the hind paws of the rat is placed on the hot plate, and the other hind paw is placed in the laboratory room temperature condition, and the tested paw withdrawal time is observed and recorded. The average value of three times measured is recorded as a basic pain threshold. If the measurement result of the paw withdrawal time exceeds 5.0 seconds, the basic pain threshold of the rat does not meet the requirements and should be excluded.

(87) 2.3 Grouping and Administration:

(88) Twenty-four qualified rats (animals with no dyskinesia and pain threshold change after catheterization) are randomly divided into 6 groups, 4 rats in each group, the first group is a control group, the experimental animals are administered with normal saline, and the other groups are administered with different doses of bupivacaine injection. The grouping information is shown in Table 1. Each group is injected with a corresponding drug through the catheter around the sciatic nerve.

(89) TABLE-US-00006 TABLE 1 Experimental animal grouping and drug administration information Admin- Actual istration Admin- Animal doses doses volume istration groups numbers drugs (mg/kg) mg/rat ml/rat mode 1 4 Vehicle — — 0.4 trans- catheter 2 4 Bupivacaine 3 0.66  0.4 trans- injection catheter group 3 4 Bupivacaine 1 0.22  0.4 trans- injection catheter group 4 4 Bupivacaine 0.3 0.066  0.4 trans- injection catheter group 5 4 Bupivacaine 0.1 0.022  0.4 trans- injection catheter group 6 4 Bupivacaine 0.03 0.0066 0.4 trans- injection catheter group *The dose is based on the actual experiment; the administration dose is determined according to the average body weight of the experimental animals. The administration concentration and volume of the animals in the single dose group are the same, for example, the animal average animal weight is 220 g, and the administration dose is 1 mg/kg, and the administration concentrations are determined to be 0.66 mg/ml, 0.4 ml/per rat.

(90) 2.4 Sensory Block (Hot Plate Paw Withdrawal Time):

(91) The sensory block of the rats is examined by a hot plate method. The hot plate is heated to (56±1)° C., one of the hind paws of the rat is placed on the hot plate, and the other hind paw is placed at room temperature in the laboratory, the tested paw withdrawal time is observed and recorded. The average value of two to three times is recorded as a pain threshold. The paw withdrawal time of the hot plate method is recorded. In order to avoid the damage of the experimental animal body, if the rat has not withdrawal paw in the hot plate experiment for more than 15 s, the rat should be removed from the hot plate, and the experimental result is recorded as 15 s. The time point for determining the paw withdrawal time of the hot plate is before administration, and 5 minutes, 10 minutes, 30 minutes, 1 hour, and 2 hours after administration.

(92) 2.5 Motor Block (Four-Level Score):

(93) The exercise condition of each group of rats is evaluated by a motor block four-level scoring method. The scoring criteria are: paws can be dorsiflexed, stretched, valgus and other normal exercises, scored by 1 point; paws can be dorsiflexed, bendable or after the adduction, although the paws can be stretched, the stretching ability is weakened, scored by 2 points; the paws can perform dorsiflexion, but no longer has the ability to stretch, scored by 3 points; the paws completely lose the ability of dorsiflexion, curling and stretching, and the rats produce a gait defect, scored by 4 points. The time point for measuring the motor block score is before administration, and 5 min, 10 min, 30 min, 1 h, and 2 h after administration.

(94) All clinical symptoms of each animal should be recorded at the beginning of the experiment and during the experiment, and observation should be performed at the same time every day.

(95) 3. Experimental Result

(96) 3.1 Clinical Symptoms

(97) In the experiment, no obvious clinical abnormal symptoms are observed in all groups of animals.

(98) 3.2 Effect of Different Doses of Bupivacaine Injection on the Hot Plate Paw Withdrawal Time of SD Rats

(99) The effect of different doses of bupivacaine injection on the hot plate paw withdrawal time of SD rats is shown in Table 2. From the data in Table 2, it can be seen:

(100) In the absence of administration, there is no significant difference in the hot plate paw withdrawal time between the experimental SD rats in each group (p>0.05).

(101) After transcatheter administration, compared with the vehicle group, the hot plate paw withdrawal time of experimental SD rats in different doses of bupivacaine injection group has different changes. Compared with the vehicle group, the hot plate paw withdrawal time of the experimental SD rats in the group of bupivacaine injection of 0.66 mg/rat is significantly higher than that of the vehicle group at the 5, 10, 30, 60, and 120 min time points (p<0.05); the hot plate paw withdrawal time of the experimental SD rats in the group of bupivacaine injection of 0.22 mg/rat is significantly higher than that of the vehicle group at the 30 and 60 min time points (p<0.05); the hot plate paw withdrawal time of the experimental SD rats in the group of bupivacaine injection of 0.066 mg/rat is significantly higher than that of the vehicle group at the 5, 10, 30, 60, and 120 min time points (p<0.05); the hot plate paw withdrawal time of the experimental SD rats in the group of bupivacaine injection of 0.022 mg/rat is significantly higher than that of the vehicle group at the 60 min time points (p<0.05); and the hot plate paw withdrawal time of the experimental SD rats in the group of bupivacaine injection of 0.00066 mg/rat is significantly higher than that of the vehicle group at the 5 and 60 min time points (p<0.05).

(102) After the solvent control group is administered with normal saline, the reduction of the hot plate paw withdrawal time of the animals is observed. According to the analysis, it may be due to the osmotic pressure and temperature of the normal saline, which causes the local stimulation to play a certain effect on the algesthesia.

(103) TABLE-US-00007 TABLE 2 Effect of different doses of bupivacaine injection on the hot plate paw withdrawal time of SD rats Groups 0 min 5 min 10 min 30 min 60 min 120 min 1 2.75 ± 1.49 1.55 ± 1.11 2.27 ± 1.46 1.52 ± 0.7  1.42 ± 0.53  1.65 ± 1.13 2 3.26 ± 0.88  6.32 ± 2.29*   7.7 ± 3.88* 7.85 ± 3.36* 5.91 ± 1.9*   4.26 ± 1.42* 3 2.75 ± 0.74 5.17 ± 5.41 7.13 ± 5.43 7.13 ± 4.01* 3.24 ± 0.85* 2.13 ± 0.8  4 3.42 ± 0.87  6.08 ± 1.68*  7.11 ± 2.55* 7.09 ± 1.45* 3.62 ± 1.25*  3.76 ± 0.69* 5 3.08 ± 0.61 4.41 ± 3.24 4.22 ± 2.45 3.59 ± 1.89  2.63 ± 0.41* 2.96 ± 1.38 6 3.06 ± 1.1   3.36 ± 0.91* 2.67 ± 0.6  3.11 ± 1.22  2.66 ± 0.53* 3.09 ± 0.82 Results are expressed as mean ± SD, *p <0.05.

(104) 3.3 Effects of Different Doses of Bupivacaine Injection on Motor Function Scores of SD Rats

(105) The effect of different doses of bupivacaine injection on the motor function score of SD rats is shown in Table 3. From the data in Table 3, it can be seen:

(106) There is no significant difference in motor function scores between the experimental SD rats in each group without drug administration.

(107) After transcatheter administration, compared with the vehicle group, the motor function score of the experimental SD rats in the group of bupivacaine injection of 0.66 mg/rat is significantly higher than that of the vehicle group at the 10, 30, and 60 min time points (p<0.05). The group of bupivacaine injection of 0.22 mg/rat is higher than the control group, but there is no statistical difference (p>0.05); the motor function scores of the experimental SD rats in the other dose groups are the same as those in the control group.

(108) TABLE-US-00008 TABLE 3 Effect of different doses of bupivacaine injection on motor function scores of SD rats Groups 0 min 5 min 10 min 30 min 60 min 120 min 1 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 2 1 ± 0 1 ± 0 1.75 ± 0.5* 1.75 ± 0.5* 1.75 ± 0.5* 1 ± 0 3 1 ± 0  1.5 ± 0.58  1.5 ± 0.58 1.25 ± 0.5  1 ± 0 1 ± 0 4 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 5 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 6 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 Results are expressed as mean ± SD, *p <0.05.

(109) Bupivacaine injection showed a motor block effect and a hot plate analgesic effect at a dose of 3 mg/kg (0.66 mg/rat) and 1 mg/kg (0.22 mg/rat). Bupivacaine injection showed a hot plate analgesic effect at doses of 0.3, 0.1, 0.03 mg/kg (ie, 0.066, 0.022, 0.066 mg/rat), but no motor nerve block effect is observed, i.e. a non-anesthetic analgesic effect is achieved.

Example 29 PEG-Bupivacaine Conjugate Hydrolysis Drug Release Experiment

(110) Preparation of isotonic phosphate buffer (0.01 mol/L, pH 7.4, PBS): 8.0 g of NaCl, 0.2 g of KH.sub.2PO.sub.4, 2.9 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.2 g of KCl are added sequentially to a quantitative container, and an appropriate amount of distilled water is added to dissolve, and then the volume is adjusted to 1000 mL and the obtained buffer is stored in a refrigerator at 4° C. for use.

(111) Hydrolysis drug release for the PEG-bupivacaine conjugate: PEG-bupivacaine products (bupivacaine conjugate 6, prepared in Example 9 of the present invention; bupivacaine conjugate 10, prepared in Example 16 of the present invention) are dissolved in PBS buffer at about 2 mg/mL and placed in a water-bathing constant temperature vibrator at 37° C. Samples are taken at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h, and the content of free bupivacaine is determined by HPLC.

(112) The experimental results are shown in Table 4. As can be seen from the data in Table 4, the hydrolysis half-life of the bupivacaine conjugate 6 is about 22 h, the hydrolysis half-life of the bupivacaine conjugate 10 is about 35 h, and the bupivacaine conjugate 10 is more stable than the bupivacaine conjugate 6. Both PEG-bupivacaine products can slowly release free bupivacaine.

(113) TABLE-US-00009 TABLE 4 Hydrolysis drug release rate of PEG-bupivacaine in pH 7.4 buffer PEG-bupivacaine hydrolysis rate(%) Time (h) bupivacaine conjugate 6 bupivacaine conjugate 10 0 0.57 0.43 6 18.58 15.03 12 33.21 25.19 24 52.85 40.37 48 76.36 61.72 72 87.12 76.25 96 92.89 87.28 120 95.02 92.76 144 95.21 96.13

Example 30 Study of Non-Anesthetic Analgesic Effect of PEG-bupivacaine Conjugate

(114) Male Sprague-Dawley rats are used in the study, about 230-250 g when accepted, and acclimated for at least 7 days prior to the experiment. Two rats are raised in each cage and fed freely with feed and water. The environmental conditions of the breeding room are 23±2° C., and the relative humidity is 40% to 70%, alternating 12 hours of light and dark. SD rats with basic pain thresholds that do not meet the requirements are excluded through screening by a hot plate method before the experiment. The qualified rats are randomly divided into 4 groups, 4 rats in each group, which are labeled as groups G1, G2, G3, and G4, respectively. Administration by direct injection in the sciatic nerve is adopted, and with the rat's back facing up, the drug is injected around the right sciatic nerve trunk between the right hip and the right thigh. The grouping information of the experimental animals is shown in Table 5.

(115) TABLE-US-00010 TABLE 5 Experimental animal grouping and drug administration information Adminis- tration Adminis- Animal Dose volume tration Groups numbers Drugs (mg/kg).sup.a ml/rat mode G1 4 Vehicle — 0.4 Direct injection G2 4 bupivacaine 32 0.4 Direct conjugate 10 injection G3 4 bupivacaine 24 0.4 Direct conjugate 11 injection G4 4 bupivacaine 48 0.4 Direct conjugate 12 injection Note: .sup.aThe dose shown refers to the dose of PEG-bupivacaine converted to bupivacaine according to the drug loading amount.

(116) The bupivacaine conjugates 10, 11 and 12 are the products prepared in the examples 16, 19 and 22 of the invention, respectively.

(117) Sensory block (hot plate paw withdrawal time): The sensory block of the rat is examined by a hot plate method, and the hot plate is heated to (56±1)° C., and one of the hind paws of the rat is placed on the hot plate. The other hind paw is in the laboratory room temperature condition, and the tested paw withdrawal time is observed and recorded. The average value of two to three times measured is recorded as a pain threshold. In order to avoid the damage of the experimental animal body, if the rat has no paw withdrawal in the hot plate experiment for more than 15 s, the rat should be removed from the hot plate, and the experimental result is recorded as 15 s. The time point for determining the paw withdrawal time of the hot plate is before administration, and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h after administration.

(118) The experimental results are shown in Table 6. As can be seen from the data in Table 6, there is no significant difference in the hot plate paw withdrawal time of SD rats in each experimental group in the absence of administration. After direct injection for administration, the hot plate paw withdrawal time of the SD rats in the experimental group of two different structures of PEG-bupivacaine and the vehicle group both are changed. The hot plate paw withdrawal time of SD rats in the group of the bupivacaine conjugate 10 of 32 mg/kg is significantly higher than that of the vehicle group at 0.5 h, 1 h, 2 h, 3 h, 4 h and 8 h after administration; the hot plate paw withdrawal time of SD rats in the group of the bupivacaine conjugate 11 of 24 mg/kg is significantly higher than that of the vehicle group within 6 h after administration; the hot plate paw withdrawal time of SD rats in the group of the bupivacaine conjugate 12 of 48 mg/kg is significantly higher than that of the vehicle group within 2 h after administration.

(119) TABLE-US-00011 TABLE 6 Effect of PEG-bupivacaine conjugate on the hot plate paw withdrawal time of SD rats Dose Groups Drugs (mg/kg) 0.5 h l h 2 h 4 h 6 h 8 h 10 h 12 h 24 h G1 Vehicle — 2.68 3.51 3.83 2.63 3.64 2.66 2.61 2.34 2.21 G2 bupivacaine 32 11.85 11.98 8.91 10.74 11.92 8.49 6.64 3.82 3.81 conjugate 10 G3 bupivacaine 24 9.60 9.99 5.02 6.65 6.44 3.69 3.24 2.62 3.77 conjugate 11 G4 bupivacaine 48 6.49 6.77 3.52 4.82 4.59 4.34 4.30 2.80 3.25 conjugate 12

(120) Motor block (four-level score): The exercise condition of each group of rats is evaluated by the motor block four-level scoring method. The scoring criteria are: paws can be dorsiflexed, stretched, valgus and other normal exercises, scored by 1 point; paws can be dorsiflexed, bendable or after the adduction, although the paws can be stretched after the adduction, the stretching ability is weakened, scored by 2 points; the paw can perform dorsiflexion, but no longer has the ability to stretch, scored by 3 points; the paws completely lose the ability of dorsiflexion, curling and stretching, and the rats produce a gait defect, scored by 4 points. The time point for measuring the motor block score is before administration, and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration.

(121) The experimental results are shown in Table 7. From the data in Table 7, it is found that there is no significant difference in motor function scores of SD rats in each experimental group in the absence of administration. After the direct injection for administration, the motor scores of bupivacaine conjugates 10, 11 and 12 at different time points are not significantly different from those of the vehicle group.

(122) From the results of sensory block and motor block, the bupivacaine conjugate 10 does not affect the motor function of SD rats when administered at 32 mg/kg, and the analgesic effect could reach 8 h; the bupivacaine conjugate 11 does not affect the motor function of SD rats when administered at 24 mg/kg, and the analgesic effect could reach 6 h.

(123) TABLE-US-00012 TABLE 7 Effect of PEG-bupivacaine conjugate on motor function score of SD rats Dose Groups Drugs (mg/kg) 0 h 0.5 h 1 h 2 h 4 h 6 h 8 h 12 h 24 h G1 Vehicle — 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 G2 bupivacaine 32 1 ± 0 1 ± 0 1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1 ± 0 1 ± 0 conjugate 10 G3 bupivacaine 24 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 conjugate 11 G4 bupivacaine 48 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 conjugate 12

Example 31 Study of Analgesic Effect of PEG-bupivacaine Conjugate on Rat Plantar Incision

(124) After SD rats inhale anesthesia (isoflurane), an incision of about 1 cm in length from the proximal end of the pelma to the toe is made: after the skin and fascia are cut, the plantar muscle is picked up with an ophthalmic forceps and cut longitudinally (maintaining the starting and ending point and attachment of muscles); pressing is carried out for hemostasis, physiological saline and test drugs are injected into the muscles of animal's legs, and then the pain threshold detection is performed by the hot plate method. Within 24 hours after the model is successful, the rats develop significant hyperalgesia. Qualified rats are randomly divided into 5 groups, 6 rats in each group, which are labeled as groups G1, G2, G3, G4 and G5, respectively. The plantar subcutaneous injection method for administration is used, and the grouping information of the experimental animals is shown in Table 8.

(125) TABLE-US-00013 TABLE 8 Experimental animal grouping and drug administration information Adminis- tration Animal Dose volume Groups numbers Drugs (mg/kg).sup.a ml/rat G1 6 Vehicle — 0.4 G2 6 bupivacaine 8 0.4 G3 6 bupivacaine conjugate 10 32 0.4 G4 6 bupivacaine conjugate 11 24 0.4 G5 6 bupivacaine conjugate 12 48 0.4 Note: .sup.aThe dose shown refers to the dose of PEG-bupivacaine converted to bupivacaine according to the drug loading amount.

(126) Sensory block (hot plate paw withdrawal time): The sensory block of the rat is examined by a hot plate method, and the hot plate is heated to (56±1)° C., and one of the hind paws of the rat is placed on the hot plate, the other hind paw is in the laboratory room temperature condition, and the tested paw withdrawal time is observed and recorded. The average value of two to three times measured is recorded as a pain threshold. In order to avoid the damage of the experimental animal body, if the rat has no paw withdrawal in the hot plate experiment for more than 15 s, the paws should be removed from the hot plate, and the experimental result is recorded as 15 s. The time point for determining the paw withdrawal time of the hot plate is before administration, and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration.

(127) The experimental results are shown in Table 9. As can be seen from the data in Table 9, there is no significant difference in the hot plate paw withdrawal time of SD rats in each experimental group in the absence of administration. After direct injection for administration, the hot plate paw withdrawal time of the SD rats in the experimental group of three different structures of PEG-bupivacaine and the vehicle group both are changed. The plantar incision model reduces the hot plate paw withdrawal time of the rats from 5.79 s before surgery to 1.23 s after surgery. The hot plate paw withdrawal time of SD rats with bupivacaine injection of 8 mg/kg is significantly higher than that of the vehicle group only at 0.5 h and 1 h after administration; the hot plate paw withdrawal time of SD rats with bupivacaine conjugate 10 injection of 32 mg/kg is significantly higher than that of the vehicle group at 0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h after administration; the hot plate paw withdrawal time of SD rats with bupivacaine conjugate 11 injection of 24 mg/kg is significantly higher than that of the vehicle group within 6 h after administration; the hot plate paw withdrawal time of SD rats with bupivacaine conjugate 12 injection of 48 mg/kg is significantly higher than that of the vehicle group within 2 h after administration.

(128) TABLE-US-00014 TABLE 9 Effect of PEG-bupivacaine conjugate on the hot plate paw withdrawal time of SD rats Base value Dose before Groups Drugs (mg/kg) surgery 0 h 0.5 h 1 h 2 h 4 h 6 h 8 h 12 h 24 h G1 Vehicle — 5.79 1.23 1.20 0.99 1.14 1.67 2.10 1.32 2.43 2.10 G2 bupivacaine 8 4.70 1.02 7.21 6.91 1.30 1.51 1.32 2.41 1.41 1.45 G3 bupivacaine 32 5.20 1.41 6.52 7.91 6.21 6.79 7.42 7.43 2.10 1.32 conjugate 10 G4 bupivacaine 24 4.97 1.26 7.43 8.49 9.10 7.91 8.65 2.19 1.89 1.56 conjugate 11 G5 bupivacaine 48 5.81 1.89 8.13 7.21 6.12 2.49 2.34 2.10 1.92 1.76 conjugate 12

(129) Motor block (four-level score): The exercise condition of each group of rats is evaluated by the motor block four-level scoring method. The scoring criteria are: paws can be dorsiflexed, stretched, valgus and other normal exercises, scored by 1 point; paws can be dorsiflexed, bendable or after the adduction, although the paws can be stretched, the stretching ability is weakened, scored by 2 points; the paws can perform dorsiflexion, but no longer has the ability to stretch, scored by 3 points; the paws completely lose the ability of dorsiflexion, curling and stretching, and the rats produce a gait defect, scored by 4 points. The time point for measuring the motor block score is before administration, and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration.

(130) The experimental results are shown in Table 10. From the data in Table 10, there is no significant difference in motor function scores of SD rats in each experimental group in the absence of administration. After direct injection for administration, from the results of sensory block and motor block, the bupivacaine conjugate 10 does not affect the motor function of SD rats when administered at 32 mg/kg, and the analgesic effect can reach 8 h; the bupivacaine conjugate 11 does not affect the motor function of SD rats when administered at 24 mg/kg, and the analgesic effect can reach 6 h or above; the bupivacaine conjugate 12 does not affect the motor function of SD rats when administered at 48 mg/kg, and the analgesic effect can reach 2 h.

(131) TABLE-US-00015 TABLE 10 Effect of PEG-bupivacaine conjugate on motor function score of SD rats Dose Groups Drugs (mg/kg) 0 h 0.5 h 1 h 2 h 4 h 6 h 8 h 12 h 24 h G1 Vehicle — 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 G2 bupivacaine 8 1 ± 0 1 ± 0 1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1 ± 0 1 ± 0 G3 bupivacaine 32 1 ± 0 1 ± 0 1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1 ± 0 1 ± 0 conjugate 10 G4 bupivacaine 24 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 conjugate 11 G5 bupivacaine 48 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 conjugate 12

Example 32 Study of Analgesic Effect of PEG-Bupivacaine Conjugate on Rat Sciatica Model

(132) Intervertebral disc compression (SNL): This model is a pathological form that simulates clinical disc herniation. The specific method is to remove the autogenous caudal intervertebral disc tissue (annulus fibrosus and nucleus pulposus) form the rats and place the caudal intervertebral disc tissue between the proximal nerve root of the L5 dorsal root ganglion and the lower vertebral body to directly compress the L5 nerve root, wherein damage to the L5 nerve root or dorsal root ganglion should be avoided during the operation. 3 to 15 days after the model is successful, rats develop significant hyperalgesia, so the administration time we chose is between 3 days and 15 days.

(133) Qualified rats are randomly divided into 5 groups, 6 rats in each group, which are labeled as groups G1, G2, G3, G4 and G5, respectively. Administration by intramuscular injection around sciatic nerve is adopted, and the grouping information of the experimental animals is shown in Table 11.

(134) TABLE-US-00016 TABLE 11 Experimental animal grouping and drug administration information Adminis- tration Animal Dose volume Groups numbers Drugs (mg/kg).sup.a ml/rat G1 6 Vehicle — 0.4 G2 6 bupivacaine 8 0.4 G3 6 bupivacaine 32 0.4 conjugate 10 G4 6 bupivacaine 24 0.4 conjugate 11 G5 6 bupivacaine 48 0.4 conjugate 12 Note: .sup.aThe dose shown refers to the dose of PEG-bupivacaine converted to bupivacaine according to the drug loading amount.

(135) Sensory block (hot plate paw withdrawal time): The sensory block of the rat is examined by a hot plate method, and the hot plate is heated to (56±1)° C., and one of the hind paws of the rat is placed on the hot plate, the other hind paw is in the laboratory room temperature condition, and the tested paw withdrawal time is observed and recorded. The average value of two to three times measured is recorded as a pain threshold. In order to avoid the damage of the experimental animal body, if the rat has no paw withdrawal in the hot plate experiment for more than 15 s, the paws should be removed from the hot plate, and the experimental result is recorded as 15 s. The time point for determining the withdrawal time of the hot plate is before administration, and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration.

(136) The experimental results are shown in Table 12. As can be seen from the data in Table 12, there is no significant difference in the hot plate paw withdrawal time of SD rats in each experimental group in the absence of administration. After direct injection for administration, the hot plate paw withdrawal time of the SD rats in the experimental group of three different structures of PEG-bupivacaine and the vehicle group is changed. SNL causes significant hyperalgesia in rats. The hot plate paw withdrawal time is reduced from 5.19 s before surgery to 1.92 s after surgery. The hot plate paw withdrawal time of SD rats with bupivacaine injection of 8 mg/kg is significantly higher than that of the vehicle group only at 0.5 h and 1 h after administration; the hot plate paw withdrawal time of SD rats with bupivacaine conjugate 10 injection of 32 mg/kg is significantly higher than that of the vehicle group at 0.5 h, 1 h, 2 h, 4 h, and 8 h after administration; the hot plate paw withdrawal time of SD rats with bupivacaine conjugate 11 injection of 24 mg/kg is significantly higher than that of the vehicle group within 6 h after administration; the hot plate paw withdrawal time of SD rats with bupivacaine conjugate 12 injection of 48 mg/kg is significantly higher than that of the vehicle group within 2 h after administration.

(137) TABLE-US-00017 TABLE 12 Effect of PEG-bupivacaine conjugate on the hot plate paw withdrawal time of SD rats Dose Before Groups Drugs (mg/kg) surgery 0 h 0.5 h 1 h 2 h 4 h 6 h 8 h 12 h 24 h G1 Vehicle — 5.19 1.92 2.13 1.90 2.34 2.71 2.19 1.93 1.80 2.67 G2 bupivacaine 8 5.40 2.28 7.39 9.22 2.81 1.89 1.78 1.95 2.43 1.72 G3 bupivacaine 32 4.98 2.41 6.52 10.12 11.43 9.83 8.10 6.91 1.96 2.18 conjugate 10 G4 bupivacaine 24 5.15 2.14 7.43 8.49 9.10 8.31 7.10 1.97 2.71 2.76 conjugate 11 G5 bupivacaine 48 5.51 1.89 8.13 7.21 6.12 2.98 2.91 2.18 1.85 2.56 conjugate 12

(138) Motor block (four-level score): The exercise condition of each group of rats is evaluated by the motor block four-level scoring method. The scoring criteria are: paws can be dorsiflexed, stretched, valgus and other normal exercises, scored by 1 point; paws can be dorsiflexed, bendable or after the adduction, although the paws can be stretched, the stretching ability is weakened, scored by 2 points; the paw can perform dorsiflexion, but no longer has the ability to stretch, scored by 3 points; the paw completely loses the ability of dorsiflexion, curling and stretching, and the rats produce a gait defect, scored by 4 points. The time point for determining the withdrawal time of the hot plate is before administration, and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration.

(139) The experimental results are shown in Table 13. From the data in Table 13, there is no significant difference in motor function scores of SD rats in each experimental group in the absence of administration. After direct injection for administration, from the results of sensory block and motor block, the bupivacaine conjugate 10 does not affect the motor function of SD rats when administered at 32 mg/kg, and the analgesic effect can reach 8 h; the bupivacaine conjugate 11 does not affect the motor function of SD rats when administered at 24 mg/kg, and the analgesic effect can reach 6 h or above; the bupivacaine conjugate 12 does not affect the motor function of SD rats when administered at 48 mg/kg, and the analgesic effect can reach 2 h.

(140) TABLE-US-00018 TABLE 13 Effect of PEG-bupivacaine conjugate on motor function score of SD rats Dose Groups Drugs (mg/kg) 0.5 h 1 h 2 h 4 h 6 h 8 h 12 h 24 h G1 Vehicle — 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 G2 bupivacaine 8 1 ± 0 1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1 ± 0 1 ± 0 G3 bupivacaine 32 1 ± 0 1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1.75 ± 0.5  1 ± 0 1 ± 0 conjugate 10 G4 bupivacaine 24 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 conjugate 11 G5 bupivacaine 48 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 conjugate 12

(141) The above embodiments are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent substitutions, etc., which are within the spirit and principles of the present invention, should be included in the protection scope of the present invention.